1. Field of the Invention
The present invention relates generally to the fields of bacterial genetics, immunology and microbial pathogenesis. More specifically, the instant invention relates to disulfide bond formation, gram-negative cell envelope structure, and protein folding and assembly in bacteria of the genus Ehrlichia. Most specifically, the present invention relates to genes encoding thio-disulfide oxidoreductases, also known as disulfide bond formation (Dsb) proteins, from Ehrlichia chaffeensis and Ehrlichia canis. 
2. Description of the Related Art
The gram-negative bacterial cell envelope consists of proteins, lipoproteins, carbohydrates and peptidoglycan, which interact to form a complex supramolecular structure. While organisms in the genus Ehrlichia have typical gram-negative cell envelope structures, limited ultrastructural studies suggest that peptidoglycan is not present. In the absence of peptidoglycan, the structure of gram-negative bacterial outer membranes may be more dependent on covalent and noncovalent associations between outer membrane proteins. Disulfide bond linkages between cell envelope proteins in Ehrlichiae have not been determined. However, covalent disulfide bonds between major surface proteins (MSPs) have been observed in the related organism Anaplasma marginale, indicating that disulfide linkages are important in the outer membrane structure (18).
Two ultrastructural forms of Ehrlichia chaffeensis, termed reticulate and dense-cored cells, correspond to ultrastructurally similar reticulate and elementary body forms observed in Chlamydiae (15). Little is known regarding the mechanism(s) of the outer membrane supramolecular rearrangements leading to these ultrastructurally defined forms, but an increase in disulfide crosslinked proteins has been described in elementary bodies of Chlamydia spp. (3). The similarity in ultrastructural forms between these two organisms indicates that disulfide bonds may be involved in cell envelope changes leading to the formation of dense-cored cells.
Thio-disulfide oxidoreductases have been characterized in the cell envelopes of several bacteria (1,5,12); these enzymes are likely to be involved in determining the three-dimensional structure of folded outer membrane proteins by catalyzing intra- and intermolecular disulfide bond formation. Although there is little overall sequence homology among disulfide bond formation proteins from various bacteria, certain features are shared, including a conserved cysteine motif (CXXC) (SEQ ID No. 1) that serves as the active site, a thioredoxin domain consisting of a secondary protein fold, and a protein reductant or oxidant activity (10,16). Such proteins have been grouped into the thioredoxin superfamily.
Disulfide oxidoreductases in Escherichia coli include thioredoxin and disulfide bond formation (Dsb) proteins A, B, C, D and E (10,16,17). DsbA and DsbB of E. coli were recognized by screening mutants that were defective in alkaline phosphatase (AP) activity, protein insertion, and sensitivity to the reducing agent dithiothreitol (DTT) (1,6,8). Other suppressors of dithiothreitol susceptibility were later identified as DsbC, D and E, enzymes that have disulfide isomerase or reductase activities (9,11). Some overlap in function occurs among these disulfide bond formation proteins, as shown by the fact that overexpression of DsbC can alleviate the defects in DsbA mutants (9).
The prior art is deficient in knowledge about the thio-disulfide oxidoreductases or disulfide bond formation proteins present in E. chaffeensis and E. canis. The present invention fulfills this longstanding need and desire in the art.